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Background and purposeThe 5-lipoxygenase product, 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), is a potent chemoattractant for eosinophils and neutrophils. However, little is known about its pathophysiological role because of the lack of a rodent ortholog of the oxoeicosanoid (OXE) receptor. The present study aimed to determine whether the selective OXE receptor antagonist S-Y048 can inhibit allergen-induced pulmonary inflammation in a monkey model of asthma.Experimental approachMonkeys sensitized to house dust mite antigen (HDM) were treated with either vehicle or S-Y048 prior to challenge with aerosolized HDM, and bronchoalveolar (BAL) fluid was collected 24 h later. After 6 weeks, animals that had initially been treated with vehicle received S-Y048 and vice versa for animals initially treated with S-Y048. Eosinophils and neutrophils in BAL and lung tissue samples were evaluated, as well as mucus-containing cells in bronchi.Key resultsHDM significantly increased the numbers of eosinophils, neutrophils, and macrophages in BAL fluid 24 h after challenge. These responses were all significantly inhibited by S-Y048, which also reduced the numbers of eosinophils and neutrophils in lung tissue 24 h after challenge with HDM. S-Y048 also significantly reduced the numbers of bronchial epithelial cells staining for mucin and MUC5AC after antigen challenge.Conclusion and implicationsThis study provides the first evidence that 5-oxo-ETE may play an important role in inducing allergen-induced pulmonary inflammation and could also be involved in regulating MUC5AC in goblet cells. OXE receptor antagonists such as S-Y048 may useful therapeutic agents in asthma and other eosinophilic as well as neutrophilic diseases.

Background and purpose5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), acting via the OXE receptor, is unique among 5-lipoxygenase products in its ability to directly induce human eosinophil migration, suggesting its involvement in eosinophilic diseases. To address this hypothesis, we synthesized selective indole-based OXE receptor antagonists. Because rodents lack an OXE receptor orthologue, we sought to determine whether these antagonists could attenuate allergen-induced skin eosinophilia in sensitized monkeys.Experimental approachIn a pilot study, cynomolgus monkeys with environmentally acquired sensitivity to Ascaris suum were treated orally with the "first-generation" OXE antagonist 230 prior to intradermal injection of 5-oxo-ETE or Ascaris extract. Eosinophils were evaluated in punch biopsy samples taken 6 or 24 hr later. We subsequently treated captive-bred rhesus monkeys sensitized to house dust mite (HDM) allergen with a more recently developed OXE antagonist, S-Y048, and evaluated its effects on dermal eosinophilia induced by either 5-oxo-ETE or HDM.Key resultsIn a pilot experiment, both 5-oxo-ETE and Ascaris extract induced dermal eosinophilia in cynomolgus monkeys, which appeared to be reduced by 230. Subsequently, we found that the related OXE antagonist S-Y048 is a highly potent inhibitor of 5-oxo-ETE-induced activation of rhesus monkey eosinophils in vitro and has a half-life in plasma of about 6 hr after oral administration. S-Y048 significantly inhibited eosinophil infiltration into the skin in response to both intradermally administered 5-oxo-ETE and HDM.Conclusions and implications5-Oxo-ETE may play an important role in allergen-induced eosinophilia. Blocking its effects with S-Y048 may provide a novel therapeutic approach for eosinophilic diseases.

In patients with cystic fibrosis (CF) and asthma, elevated levels of interleukin-8 (IL-8) are found in the airways. IL-8 is a CXC chemokine that is a chemoattractant for neutrophils through CXCR1 and CXCR2 G protein-coupled receptors. We hypothesized that IL-8 acts directly on airway smooth muscle cells (ASMC) in a way that may contribute to the enhanced airway responsiveness and airway remodeling observed in CF and asthma. The aim of this study was to determine whether human ASMC (HASMC) express functional IL-8 receptors (CXCR1 and CXCR2) linked to cell contraction and migration. Experiments were conducted on cells harvested from human lung specimens. Real-time PCR and fluorescence-activated cell sorting analysis showed that HASMC expressed mRNA and protein for both CXCR1 and CXCR2. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) increased from 115 to 170 nM in response to IL-8 (100 nM) and decreased after inhibition of phospholipase C (PLC) with U-73122. On blocking the receptors with specific neutralizing antibodies, changes in [[[Ca.sup.2+]].sub.i] were abrogated. IL-8 also contracted the HASMC, decreasing the length of cells by 15%, and induced a 2.5-fold increase in migration. These results indicate that HASMC constitutively express functional CXCR1 and CXCR2 that mediate IL-8-triggered [Ca.sup.2+] release, contraction, and migration. These data suggest a potential role for IL-8 in causing abnormal airway structure and function in asthma and CF. chemokines; lung; signal transduction

5-Oxo-ETE regulates tone of guinea pig airway smooth muscle via activation of [Ca.sup.2+] pools and Rho-kinase pathway. Am J Physiol Lung Cell Mol Physiol 287: L631-L640, 2004. First published April 16, 2004: 10.l152/ajplung.00005.2004.--5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a proinflammatory mediator, but its effects on airway smooth muscle (ASM) have never been assessed. Tension measurements performed on guinea pig ASM showed that 5-oxo-ETE induced sustained concentration-dependent positive inotropic responses (E[C.sub.50] = 0.89 [micro]M) of somewhat lower amplitude than those induced by carbamylcholine and the thromboxane [A.sub.2] (TX[A.sub.2]) agonist U-46619. Transient inotropic responses to 5-oxo-ETE were recorded in [Ca.sup.2+]-free medium, suggesting mobilization of intracellular [Ca.sup.2+]. Meanwhile, the sustained contraction, which required [Ca.sup.2+] entry, was partially blocked by I [micro]M nifedipine (an L-type [Ca.sup.2+] channel blocker) but relatively insensitive to 100 [micro]M [Gd.sup.3+]. The 5-oxo-ETE responses were also inhibited by indomethacin and SC-560 [a cyclo-oxygenase (COX-1) inhibitor] pretreatments but not by NS-398 (a selective COX-2 inhibitor). The contractile effects of 5-oxo-ETE on ASM were inhibited by the selective TX[A.sub.2] receptor (TP receptor) antagonist SQ-29548 (-75%) and by 2-(p-amylcinnamoyl) amino-4-chiorobenzoic acid pretreatment, a phospholipase [A.sub.2] inhibitor (-66%), suggesting that the major part of its effect is mediated by the release of TX[A.sub.2]. ASM responses to 5-oxo-ETE were also blocked by the Rho-kinase inhibitor Y-27632, which also partially inhibited the response to the TP receptor agonist U-46619, suggesting that the contractile response is due in part to [Ca.sup.2+] sensitization of ASM cell myofilaments. 5-oxo-eicosatetraenoic acid; isometric tension: membrane potential; calcium entry; transient receptor potential; Rho-kinase

A study relating to research on the biological activity of oxoeicosanoids describes the first total synthesis of 5-oxo-12(S)-hydroxy-6(E),8(Z),10(E), 14(Z)-eicosatetraenoic acid and its 8,9-trans-isomer and their identification in human platelets. These synthetic materials were used to determine their formation by mixtures of platelets and neutrophils as a result of a trans-cellular metabolism between neutrophils and platelets.

A study was conducted to characterize the synthesis of 11,12,14,15-tetratritiated and deuterated 5-oxo-ETE using a new bisacetylene precursor. Synthetic 5-oxo-ETE was utilized to determine the structure of biologically-derived mediators. A modified hydrogenation and deuteration experiment was carried out to determine the tritiation of a bisacetylene derivative. Results showed that catalysts can be transferred during filtration through Celite.

A novel design for template-selective recognition sites in polymers prepared by molecular imprinting is reported. Molecular imprints were prepared against cAMP that contain a fluorescent dye, trans-4-[p-(N,N-dimethylamino)styryl]-N-vinylbenzylpyridinium chloride, as an integral part of the recognition cavity, thus serving as both the recognition element and the measuring element for the fluorescence detection of cAMP in aqueous media. This fluorescent molecularly imprinted polymer displays a quenching of fluorescence in the presence of aqueous cAMP, whereas almost no effect is observed in the presence of the structurally similar molecule, cGMP. The association constant for the binding of cAMP to the imprinted polymer was determined to be in the order of [10.sup.5] [M.sup.-1]. Such fluorescent molecularly imprinted polymers containing selective sites for cAMP may find applications as fluorescent chemosensors for the aqueous detection of this molecule.

A study was conducted on the total synthesis of a potent inflammatory mediator 5-oxo-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid and its biotransformation product 6,7-dihydro-5-oxo-ETE. The findings indicate that the 6,7-dihydro derivative is about 100 times less potent than 5-oxo-ETE in stimulating cytosolic calcium immobilization in human neutrophils. Thus, the reduction of this double bond may contribute to the biological inactivation of 5-oxo-ETE.

The first total synthesis of 10,11-dihydro-12-oxo-LTB4, together with its 6,7-trans isomer, is described. The synthesis proceeds with the preparation of octenyl dithiane from nonenol in a two-step one-pot reaction and its alkylation with 2-(2-bromoethyl)-1,3-dioxolane to produce dioxolane. Hydrolysis of the dioxolane yielded aldehyde, which was reacted with trimethyl phosphonoacetate with LDA as base to produce a mixture of E- and Z-isomers. 10,11-Dihydro-12-oxo-LTB4 was found to be a major pivotal intermediate in the biotransformation of leukotriene B4 by the 'LTB4 reductase pathway.'

The very unstable biochemical intermediate, 5(S)-hydroxy-12-oxo-6(Z),8(E),10(E),14(Z)-eicosatetraenoic acid is synthesized by artificial methods. The synthesis is performed by the incubation of 12-oxo-LTB4 with human keratinocytes along with two new intriguing mediators. The synthesized LTB is compared with other similar compounds and its properties are determined.

Human polymorphonuclear leukocytes (PMNL), which severely affect the level of cytosolic calcium in leukocytic cells, chemotactically react with the newly synthesized compound 5-oxo-6,8,11,14-eicosatetraenoic acid. The compound is synthesized from 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) using a particular microsomal dehydrogenase. The reaction produced only a very low amount of 5-HETE.

Abstract Background Exposure to chlorine (Cl2) causes airway injury, characterized by oxidative damage, an influx of inflammatory cells and airway hyperresponsiveness. We hypothesized that Cl2-induced airway injury may be attenuated by antioxidant treatment, even after the initial injury. Methods Balb/C mice were exposed to Cl2 gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl2, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl2 exposure. Results Mice exposed to Cl2 had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl2 exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl2-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl2 prevented lipid peroxidation in the lung. Following Cl2 exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl2 exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU. Conclusion Our data show that the anti-oxidant DMTU is effective in attenuating Cl2 induced increase in airway responsiveness, inflammation and biomarkers of oxidative stress.